Mutations in the NUP160 gene, which encodes one protein component of the nuclear pore complex nucleoporin 160 kD, are implicated in steroid-resistant nephrotic syndrome, an international team reports March 25, 2019, in the Journal of the American Society of Nephrology. Mutations in this gene have not been associated with steroid-resistant nephrotic syndrome previously.

“Our findings indicate that NUP160 should be included in the gene panel used to diagnose steroid-resistant nephrotic syndrome to identify additional patients with homozygous or compound-heterozygous NUP160 mutations,” says Zhe Han, Ph.D., an associate professor in the Center for Genetic Medicine Research at Children’s National and the study’s senior author.

The kidneys filter blood and ferry waste out of the body via urine. Nephrotic syndrome is a kidney disease caused by disruption of the glomerular filtration barrier, permitting a significant amount of protein to leak into the urine. While some types of nephrotic syndrome can be treated with steroids, the form of the disease that is triggered by genetic mutations does not respond to steroids.

The patient covered in the JASN article had experienced persistently high levels of protein in the urine (proteinuria) from the time she was 7. By age 10, she was admitted to a Shanghai hospital and underwent her first renal biopsy, which showed some kidney damage. Three years later, she had a second renal biopsy showing more pronounced kidney disease. Treatment with the steroid prednisone; cyclophosphamide, a chemotherapy drug; and tripterygium wilfordii glycoside, a traditional therapy, all failed. By age 15, the girl’s condition had worsened and she had end stage renal disease, the last of five stages of chronic kidney disease.

An older brother and older sister had steroid-resistant nephrotic syndrome as well and both died from end stage kidney disease before reaching 17. When she was 16, the girl was able to receive a kidney transplant that saved her life.

Han learned about the family while presenting research findings in China. An attendee of his session said that he suspected an unknown mutation might be responsible for steroid-resistant nephrotic syndrome in this family, and he invited Han to work in collaboration to solve the genetic mystery.

By conducting whole exome sequencing of surviving family members, the research team found that the mother and father each carry one mutated copy of NUP160 and one good copy. Their children inherited one mutated copy from either parent, the variant E803K from the father and the variant R1173X, which causes truncated proteins, from the mother. The woman (now 29) did not have any mutations in genes known to be associated with steroid-resistant nephrotic syndrome.

Some 50 different genes that serve vital roles – including encoding components of the slit diaphragm, actin cytoskeleton proteins and nucleoporins, building blocks of the nuclear pore complex – can trigger steroid-resistant nephrotic syndrome when mutated.

With dozens of possible suspects, they narrowed the list to six variant genes by analyzing minor allele frequency, mutation type, clinical characteristics and other factors.

Significantly, the dramatic structural and functional defects caused by silencing of fly Nup160 gene in nephrocytes could be completely rescued by expressing the wild-type human NUP160 gene, but not by expressing the human NUP160 gene carrying the E803K or R1173X mutation identified from the girl’s family.

“This study identified new genetic mutations that could lead to steroid-resistant nephrotic syndrome,” Han notes. “In addition, it demonstrates a highly efficient Drosophila-based disease variant functional study system. We call it the ‘Gene Replacement’ system since it replaces a fly gene with a human gene. By comparing the function of the wild-type human gene versus mutant alleles from patients, we could determine exactly how a specific mutation affects the function of a human gene in the context of relevant tissues or cell types. Because of the low cost and high efficiency of the Drosophila system, we can quickly provide much-needed functional data for novel disease-causing genetic variants using this approach.”

Financial support for research described in this post was provided by the Nature Science Foundation of Fujian Province of China, under grant 2015J01407; National Nature Science Foundation of China, under grant 81270766; Key Project of Social Development of Fujian Province of China, under grant 2013Y0072; and the National Institutes of Health, under grants DK098410 and HL134940.

A Children’s researcher has received a $2 million grant from the National Institutes of Health (NIH) to study nephrotic syndrome in Drosophila, a basic model system that has revealed groundbreaking insights into human health. The award for Zhe Han, Ph.D., an associate professor in Children’s Center for Genetic Medicine Research, is believed to be the first ever NIH Research Project grant (R01) to investigate glomerular kidney disease using Drosophila. Nephrotic syndrome is mostly caused by damage of glomeruli, so it is equivalent to glomerular kidney disease.

“Children’s National leads the world in using Drosophila to model human kidney diseases,” Han says.

In order to qualify for the five-year funding renewal, Han’s lab needed to successfully accomplish the aims of its first five years of NIH funding. During the first phase of funding, Han established that nephrocytes in Drosophila serve the same functions as glomeruli in humans, and his lab created a series of fly models that are relevant for human glomerular disease.

“Some 85 percent of the genes known to be involved in nephrotic syndrome are conserved from the fly to humans. They play similar roles in the nephrocyte as they play in the podocytes in human kidneys,” he adds.

Pediatric nephrotic syndrome is a constellation of symptoms that indicate when children’s kidneys are damaged, especially the glomeruli, units within the kidney that filter blood. Babies as young as 1 year old can suffer proteinuria, which is characterized by too much protein being released from the blood into the urine.

“It’s a serious disease and can be triggered by environmental factors, taking certain prescription medicines or inflammation, among other factors. Right now, that type of nephrotic syndrome is mainly treated by steroids, and the steroid treatment works in many cases,” he says.

However, steroid-resistant nephrotic syndrome occurs primarily due to genetic mutations that affect the kidney’s filtration system: These filters are either broken or the protein reabsorption mechanism is disrupted.

“When genetics is to blame, we cannot turn to steroids. Right now there is no treatment. And many of these children are too young to be considered for a kidney transplant,” he adds. “We have to understand exactly which genetic mutation caused the disease in order to develop a targeted treatment.”

With the new funding, Han will examine a large array of genetic mutations that cause nephrotic syndrome. He’s focusing his efforts on genes involved in the cytoskeleton, a network of filaments and tubules in the cytoplasm of living cells that help them to maintain shape and carry out important functions.

“Right now, we don’t really understand the cytoskeleton of podocytes – highly specialized cells that wrap around the capillaries of the glomerulus – because podocytes are difficult to access. To change a gene requires time and considerable effort in other experimental models. However, changing genes in Drosophila is very easy, quick and inexpensive. We can examine hundreds of genes involving the cytoskeleton and see how changing those genes affect kidney cell function,” he says.

Han’s lab already found that Coenzyme Q10, one of the best-selling nutrient supplements to support heart health also could be beneficial for kidney health. For the cytoskeleton, he has a different targeted medicine in mind to determine whether Rho inhibitors also could be beneficial for kidney health for patients with certain genetic mutations affecting their podocyte cytoskeleton.

“One particular aim of our research is to use the same strategy as we employed for the Coq2 gene to generate a personalized fly model for patients with cytoskeleton gene mutations and test potential target drugs, such as Rho inhibitors.” Han added. “As far as I understand, this is where the future of medicine is headed.”

Dr. Moxey-Mims and two additional scientific advisers were selected for their commitment to improving care for patients with glomerular disease, diseases that impair kidney function by attacking blood cleaning units within the kidney.

During her tenure at the National Institute of Diabetes and Digestive and Kidney Diseases at the National Institutes of Health, Dr. Moxey-Mims launched the Chronic Kidney Disease in Children Cohort Study, a prospective study to investigate chronic kidney disease risk factors and outcomes, and helped launch the Cure Glomerulonephropathy Network, a multi-site study with the overarching aim to advance the diagnosis and care of patients with four different glomerular diseases.

“I am truly honored to join this distinguished group of scientific advisers and look forward to leveraging our combined strengths and research knowledge in order to deliver cures for kidney diseases faster,” says Dr. Moxey-Mims.

Mitochondria are the cell’s powerplants and inside them the MTP enzymatic complex catalyzes three steps in beta-oxidation of long-chain fatty acids.

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In one family, genetic lightning struck twice. Two sisters were diagnosed with mitochondrial trifunctional protein (MTP) deficiency. This is a rare condition that stops the body from converting fats to energy, which can lead to lactic acidosis, recurrent breakdown of muscle tissue and release into the bloodstream (rhabdomyolysis), enlarged heart (cardiomyopathy) and liver failure.

Mitochondria are the cell’s powerplants and inside them the MTP enzymatic complex catalyzes three steps in beta-oxidation of long-chain fatty acids. MTP deficiency is so rare that fewer than 100 cases have been reported in the literature says Hostensia Beng, M.D., who presented an MTP case study during the American Society of Nephrology’s Kidney Week.

The 7-month-old girl with known MTP deficiency arrived at Children’s National lethargic with poor appetite. Her laboratory results showed a low corrected serum calcium level, elevated CK level and protein in the urine (proteinuria) at a nephrotic range. The infant was treated for primary hypoparathyroidism and rhabdomyolysis.

Even though the rhabdomyolysis got better, the excess protein in the girl’s urine remained at worrisome levels. A renal biopsy showed minimal change disease and foot process fusion. And electron microscopy revealed shrunken, dense mitochondria in visceral epithelial cells and endothelium.

“We gave her tacrolimus, a calcineurin inhibitor that we are well familiar with because we use it after transplants to ensure patient’s bodies don’t reject the donated organ. By eight months after treatment, the girl’s urine protein-to-creatinine (uPCR) ratio was back to normal. At 35 months, that key uPCR measure rose again when tacrolimus was discontinued. When treatment began again, uPCR was restored to normal levels one month later,” Dr. Beng says.

The girl’s older sister also shares the heterozygous deletion in the HADHB gene, which provides instructions for making MTP. That missing section of the genetic how-to guide was predicted to cause truncation and loss of long-chain-3-hydroxyacl CoA dehydrogenase function leading to MTP deficiency.

“Renal involvement has been reported in only one patient with MTP deficiency to date, the older sister of our patient,” Dr. Beng adds.

Podocytes are specialized cells in the kidneys that provide a barrier, preventing plasma proteins from leaking into the urine. Podocytes, however, need energy to function and are rich in mitochondria.

“The proteinuria in these two sisters may be related to their mitochondrial dysfunction. Calcineurin inhibitors like tacrolimus have been reported to reduce proteinuria by stabilizing the podocyte actin cytoskeleton. Tacrolimus was an effective treatment for our patient, who has maintained normal renal function, unlike her sister,” Dr. Beng says.

A research team was able to “rescue” phenotypes caused by silencing the fly CoQ2 gene by providing nephrocytes with a normal human CoQ2 gene, as well as by providing flies with Q10, a popular supplement.

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A new study led by Children’s National research scientists shows that coenzyme Q10 (CoQ10), a popular over-the-counter supplement sold for pennies a dose, could alleviate genetic problems that affect kidney function. The work, done in genetically modified fruit flies — a common model for human genetic diseases since people and fruit flies share a majority of genes — could give hope to human patients with problems in the same genetic pathway.

The new study, published April 20 by Journal of the American Society of Nephrology, focuses on genes the fly uses to create CoQ10.

“Transgenic Drosophila that carry mutations in this critical pathway are a clinically relevant model to shed light on the genetic mutations that underlie severe kidney disease in humans, and they could be instrumental for testing novel therapies for rare diseases, such as focal segmental glomerulosclerosis (FSGS), that currently lack treatment options,” says Zhe Han, Ph.D., principal investigator and associate professor in the Center for Cancer & Immunology Research at Children’s National and senior study author.

Nephrotic syndrome (NS) is a cluster of symptoms that signal kidney damage, including excess protein in the urine, low protein levels in blood, swelling and elevated cholesterol. The version of NS that is resistant to steroids is a major cause of end stage renal disease. Of the more than 40 genes that cause genetic kidney disease, the research team concentrated on mutations in genes involved in the biosynthesis of CoQ10, an important antioxidant that protects the cell against damage from reactive oxygen.

Drosophila pericardial nephrocytes perform renal cell functions including filtering of hemolymph (the fly’s version of blood), recycling of low molecular weight proteins and sequestration of filtered toxins. Nephrocytes closely resemble, in structure and function, the podocytes of the human kidney. The research team tailor-made a Drosophila model to perform the first systematic in vivo study to assess the roles of CoQ10 pathway genes in renal cell health and kidney function.

One by one, they silenced the function of all CoQ genes in nephrocytes. If any individual gene’s function was silenced, fruit flies died prematurely. But silencing three specific genes in the pathway associated with NS in humans – Coq2, Coq6 and Coq8 – resulted in abnormal localization of slit diaphragm structures, the most important of the kidney’s three filtration layers; collapse of membrane channel networks surrounding the cell; and increased numbers of abnormal mitochondria with deformed inner membrane structure.

The flies also experienced a nearly three-fold increase in levels of reactive oxygen, which the study authors say is a sufficient degree of oxidative stress to cause cellular injury and to impair function – especially to the mitochondrial inner membrane. Cells rely on properly functioning mitochondria, the cell’s powerhouse, to convert energy from food into a useful form. Impaired mitochondrial structure is linked to pathogenic kidney disease.

The research team was able to “rescue” phenotypes caused by silencing the fly CoQ2 gene by providing nephrocytes with a normal human CoQ2 gene, as well as by providing flies with Q10, a readily available dietary supplement. Conversely, a mutant human CoQ2 gene from an patient with FSGS failed to rescue, providing evidence in support of that particular CoQ2 gene mutation causing the FSGS. The finding also indicated that the patient could benefit from Q10 supplementation.

“This represents a benchmark for precision medicine,” Han adds. “Our gene-replacement approach silenced the fly homolog in the tissue of interest – here, the kidney cells – and provided a human gene to supply the silenced function. When we use a human gene carrying a mutation from a patient for this assay, we can discover precisely how a specific mutation – in many cases only a single amino acid change – might lead to severe disease. We can then use this personalized fly model, carrying a patient-derived mutation, to perform drug testing and screening to find and test potential treatments. This is how I envision using the fruit fly to facilitate precision medicine.”

Zhe Han, Ph.D., has found that a majority of human genes known to be associated with nephrotic syndrome play conserved roles in renal function, from fruit flies to humans.

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Drosophila melanogaster, the common fruit fly, has played a key role in genetic research for decades. Even though D. melanogaster and humans look vastly different, researchers estimate that about 75 percent of human disease-causing genes have a functional homolog in the fly.

A Children’s National Health System research team reported in a recent issue of Human Molecular Genetics that the majority of genes associated with nephrotic syndrome (NS) in humans also play pivotal roles in Drosophila renal function, a conservation of function across species that validates transgenic flies as ideal pre-clinical models to improve understanding of human disease.

NS is a cluster of symptoms that signal kidney damage, including excess protein in urine, low protein levels in blood, elevated cholesterol and swelling. Research teams have identified mutations in more than 40 genes that cause genetic kidney disease, but knowledge gaps remain in understanding the precise roles that specific genes play in kidney cell biology and renal disease. To address those research gaps, Zhe Han, Ph.D., a principal investigator and associate professor in the Center for Cancer & Immunology Research at Children’s National, and colleagues systematically studied NS-associated genes in the Drosophila model, including seven genes whose renal function had never been analyzed in a pre-clinical model.

“Eighty-five percent of these genes are required for nephrocyte function, suggesting that a majority of human genes known to be associated with NS play conserved roles in renal function from flies to humans,” says Han, the paper’s senior author. “To hone in on functional conservation, we focused on Cindr, the fly’s version of the human NS gene, CD2AP,” Han adds. “Silencing Cindr in nephrocytes led to dramatic impairments in nephrocyte function, shortened their life span, collapsed nephrocyte lacunar channels – the fly’s nutrient circulatory system – and effaced nephrocyte slit diaphragms, which diminished filtration function.”

And, to confirm that the phenotypes they were studying truly caused human disease, they reversed the damage by expressing a wild-type human CD2AP gene. A mutant allele derived from a patient with CD2AP-associated NS did not rescue the phenotypes.

Thus, the Drosophila nephrocyte can be used to explain the clinically relevant molecular mechanisms underlying the pathogenesis of most monogenic forms of NS, the research team concludes. “This is a landmark paper for using the fly to study genetic kidney diseases,” Han adds. “For the first time, we realized that the functions of essential kidney genes could be so similar from the flies to humans.”

A logical next step will be to generate personalized in vivo models of genetic renal diseases bearing patient-specific mutations, Han says. These in vivo models can be used for drug screens to identify treatments for kidney diseases that currently lack therapeutic options, such as most of the 40 genes studies in this paper as well as the APOL1 gene that is associated with the higher risk of kidney diseases among millions of African Americans.

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